A number of biological responses are regulated by three component signaling pathways where a GTP-binding protein (G protein) serves as a transducer that mediates the actions of a cell surface receptor on an effector enzyme or an ion channel. One such biological response is phototransduction, which is the outcome of the actions of a light receptor, rhodopsin, a heterotrimeric G protein, transducin, and an effector enzyme, the cyclic GMP phosphodiesterase (PDE). The applicants have used the vertebrate vision system as a model to probe the molecular mechanisms underlying receptor/G protein-coupled signalling in detail. To do this key have developed a number of fluorescence spectroscopic read-outs for each of the key steps in the signal transduction pathway, including the binding of rhodopsin to the transducin-bet-gamma subunit complex (Bgr), the binding of the transducin-alpha PDE. It is their intention to utilize these read-outs for each of the key steps in the signal transduction pathway, including the binding of rhodopsin to the transducin-beta-gamma subunit complex (Bgr), the binding of the transducin-alpha subunit (At) to Bgr, and the interactions of an activated. At subunit with the PDE. It is their intention to utilize these read-outs, in combination with reconstitution and molecular biology-based approaches to address specific aspects of the functional coupling of G protein-alpha and beta-gamma subunits to receptor and effector proteins. Four avenues of investigation will constitute the Specific Aims of this proposal. These are: (1) Characterization of the individual steps of the GTP-binding/GTPase cycle of a G protein; (2) Structure-function characterization of the transducin-alpha subunit; (3) Structure-function characterization of the transducin-beta-gamma subunit complex; and (4) Determination of the molecular mechanisms underlying the interactions of the transducin-alpha subunit with the cyclic GMP PDE. Among the specific issues that will be addressed include comparisons of the rates for key steps in the activation/deactivation cycle of a G protein, an examination of the roles of receptor and/or G protein oligomerization in the G protein-activation event, the characterization of GTP-induced conformational changes in the alpha-T subunit, and the identification of the specific regions of the alpha-T subunit that are involved in binding to rhodopsin, Bgr. Other questions include what is the molecular basis by which beta-gamma complexes promote receptor/G protein-alpha coupling; what are the mechanisms underlying the stimulation of the cyclic GMP phosphodiesterase (PDE) by the alpha-T-GTP species and alpha-T? These studies will be performed in well defined reconstituted systems containing the purified signaling components of the phototransduction system and should provide detailed information regarding each step in a receptor/G protein-coupled signaling cascade.